The first step to a PCR is to identify what segment of DNA you wish to amplify. For research, one is often amplifying a single gene of interest in the study. For my current work, I use primers that are designed to identify the GUS gene of my transformed plants. This lets me identify which plants have been successfully transformed as they will have the gene while non-transformed plants will not. Picking your primers is one of the most crucial steps in PCR. Primers are small segments of DNA that will pair with the genome DNA and direct the amplification of your gene. They must be designed so that they can only base pair in your gene of interest and not to other random segments of DNA, otherwise your PCR will give you meaningless information.
Once you have your primers and your DNA sample, then comes the master mix. The master mix will contain the primers, buffer, magnesium ions, dNTPS (extra nucleotides), and a polymerase. The one we use is hot start Taq. DNA Polymerase is the enzyme that builds new DNA. Taq stands for the species from which this polymerase comes from the bacteria Thermus aquaticus. T. aquaticus lives in hot springs and thus its enzymes do not degrade under increased temperature, like those in the cycling steps of PCR.
|Our thermocycler, an oldie but goodie|
|Loaded agarose gel|
|Effect of cycle number on PCR products. |
L to R 20, 22, 25, 28, 30 cycles.